<p>HMG-CoA reductase from Pseudomonas mevalonii (<i>Pm</i>HMGR) catalyzes the oxidation of mevalonate and mevaldyl-CoA to form HMG-CoA using CoA-SH and two NAD+ cofactors. While the enzyme has been used extensively as a drug target in humans to treat hypercholesterolemia, its pathway has also been found to be critical for the survival of antibiotic resistant gram-positive bacteria. Structural studies using non-productive and slow-substrate binary complexes as well as biochemical studies using half and full reactions led to the proposal that the conversion of mevalonate to HMG-CoA occurs through the generation of two intermediates, mevaldehyde and mevaldyl-CoA (Shown in Fig 1.1). However, several intermediary changes along the <i>Pm</i>HMGR reaction pathway remain unclear. By gathering information about the enzyme’s intermediate states via structural studies, we could identify potential allosteric sites that further the reaction mechanism. Using this knowledge, we could design enzyme inhibitors that act as novel antibacterials. The application of time-resolved crystallographic methods would provide structural information about transitory states in the PmHMGR reaction mechanism. The <i>Pm</i>HMGR crystal has been shown to be suitable for time-resolved crystallographic measurements for the reaction steps resulting in mevaldyl-CoA formation. However, our structural investigations of the mevalonate, CoA and NAD+ complex that are expected to result in the formation of mevaldehyde (Fig 1.1) do not show any changes corresponding to a turnover in the crystal environment. <br></p><p><br></p><p>To investigate the factors limiting enzymatic activity in the crystal, we investigated the effects of pH and specific ions in the crystallization environment. Kinetic studies indicated a strong <i>Pm</i>HMHGR inhibition in the crystallization buffer that is dependent on the concentration of the crystallization precipitant ammonium sulfate. These studies also indicated an increase in enzyme turnover with increasing pH. Utilizing the ionic concentration and pH-dependent properties of the enzyme in the crystallization environment, we have developed a reaction triggering approach using pH changes for <i>Pm</i>HMGR crystals.<br></p><p><br></p><p>We have demonstrated our application of this ‘pH-jump’ method by observing changes in <i>Pm</i>HMGR crystals after reaction initiation. Changes in the density of mevalonate, CoA and NAD+have indicated mevaldehyde and mevaldyl-CoA formation. Additionally, the appearance of a unique NADH absorbance peak after the pH-change has also highlighted the initiation of the <i>Pm</i>HMGR reaction and the occurrence of a hydride transfer step. Our analysis of the movements using time-resolved structures post reaction-initiation have also highlighted structural changes and inter-domain contacts in the small and flap domain that would allow cofactor exchange and product release. The pH-jump method can hence be utilized as a novel approach for triggering the <i>Pm</i>HMGR reaction in crystals and further studying transitory states along its reaction pathway.<br></p>

Successful enzyme catalysis depends on many noncovalent interactions between the enzyme, cofactors, and substrate that poise the system to access a productive transition state. Motions on a variety of timescales contribute to this, but some controversy exists surrounding the role of ultrafast dynamics on catalysis. Site-specific 2D IR spectroscopy using probes of vibrational dynamics provides the opportunity to explore ultrafast motions in an enzyme active site owing to the technique’s spatial and temporal resolution. In this work, a series of aryl-azide vibrational labels were assessed using a variety of 2D IR techniques for their sensitivity to solvent and energy transfer processes, and their ability to be adapted to experiments in biomacromolecules. One of these labels, 4-azido-N-phenylmaleimide, is a substrate analog for the promiscuous ene-reductase from Pyrococcus horikoshii (PhENR). The label was covalently attached in two orientations in the enzyme active site, occupying the same position as native substrates based on X-ray crystallography and molecular dynamics simulations. FTIR and 2D IR spectroscopy were used to identify close-lying conformational states based on the strong anharmonic coupling of the label, revealing that the active site itself modulates the probe’s internal vibrational coupling. More commonly used analogous aryl-nitrile labels, however, were not sensitive to such small structural and lineshape changes. This demonstrates the importance of thoughtful label design to maximize the amount of information that can be gleaned from 2D IR studies. Using the methods herein—both spectroscopic and biochemical—provides a strategy for probing ultrafast motions that could possibly be catalytically relevant.

No description available.

<p>The rise of bacterial infections and increase of antibiotic resistant bacteria has become a major problem in the treatment of bacterial infections. The use and overuse of antibiotics, and the inherent ability of bacteria to adapt to their environment, have lead to the emergence of strains that are resistant to all antibiotics. Ideally, new targets for antibacterial drug therapy would be essential to the virulence of most or all bacteria. That is, antibiotics exploiting these targets would have broad spectrum activity. 3-Deoxy-D-arabinoheptulosonate-7- phosphate (DAHP) synthase could be such a target. This enzyme catalyzes the condensation of erythrose 4-phosphate (E4P) and phoshoenolpyruvate (PEP) to form DAHP. The DAHP synthase-catalyzed reaction is the first committed step in the shikimic acid biosynthetic pathway leading to the aromatic amino acids and other secondary metabolites in all bacteria and some parasites. Inhibition of this enzyme would lead to a depletion of aromatic amino acids within the cell, halting new protein synthesis and killing the cells. Our lab has developed a transition state analogue, DAHP oxime, which is a slow binding, potent inhibitor of DAHP synthase. Kinetic characterization of inhibitor binding revealed DAHP oxime to be a competitive inhibitor with an ultimate Ki* of 81 nM. Crystal structures of DAHP oxime bound to DAHP synthase revealed that the inhibitor bound to two of the four subunits. The two unbound subunits remain catalytically competent, suggesting that DAHP synthase may utilize a half-of-sites mechanism during catalysis. We further investigated changes in DAHP synthase dynamics in response to PEP and DAHP oxime binding via solvent hydrogen/deuterium exchange mass spectrometry. DAHP synthase in the unbound form was loosely structured around the surface exposed regions, whereas the X-ray crystal structures appeared to be more fully structured. Binding of both PEP and DAHP oxime introduced different degrees of dynamic stabilization.</p> / Doctor of Philosophy (PhD)

Pin1 is an enzyme central to cell signaling pathways because it catalyzes the cis–trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. This regulatory function makes Pin1 a drug target in the treatment of various diseases. The effects of phosphorylation on Pin1 substrates and the basis for Pin1 recognition are not well understood. The conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 were determined using molecular dynamics simulations. Phosphorylation perturbs the backbone conformational space of Pin1 substrate analogues. It is also shown that Pin1 recognizes specific conformations of its substrate by conformational selection. Dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme–substrate complex when the substrate is in the transition state configuration. This suggests that these motions play a significant role during catalysis. These results provide a detailed mechanistic understanding of Pin1 substrate recognition that can be exploited for drug design purposes and further our understanding of the subtleties of post-translational phosphorylation and cis–trans isomerization. Results from accelerated molecular dynamics simulations indicate that catalysis occurs along a restricted path of the backbone configuration of the substrate, selecting specific subpopulations of the conformational space of the substrate in the active site of Pin1. The simulations show that the enzyme–substrate interactions are coupled to the state of the prolyl peptide bond during catalysis. The transition-state configuration of the substrate binds better than the cis and trans states to the catalytic domain of Pin1. This suggests that Pin1 catalyzes its substrate by noncovalently stabilizing the transition state. These results suggest an atomistic detail understanding of the catalytic mechanism of Pin1 that is necessary for the design of novel inhibitors and the treatment of several diseases. Additionally, a set of constant force biased molecular dynamics simulations are presented to explore the kinetic properties of a Pin1 substrate and its unphosphorylated analogue. The simulations indicate that the phosphorylated Pin1 substrate isomerizes slower than the unphosphorylated analogue. This is due to the lower diffusion constant for the phosphorylated Pin1 substrate.

No description available.

No description available.

The respective kinetics of bacterial leucine aminopeptidase and [beta]-glucosidase activities were investigated to improve understanding of factors controlling activity and hydrolytic capacity in estuarine organic-poor sands. Depth distributions of enzyme activity and bulk organic matter content were determined in sediments of Aransas Bay and Copano Bay Texas, to investigate enzyme dynamics as related to the geochemical properties of the sediment. Vertical profiles of activity in sediment showed that the enzymes were more active at the surface and that the potential hydrolysis rate of leucine aminopeptidase was higher than that of [beta]-glucosidase. Vertical patterns of enzyme activity correlated (weakly) with variations in sediment organic matter (TOC, TN, and carbohydrates) content. Enrichments of sediment samples with monomeric organic compounds and inorganic nutrients did not affect leucine aminopeptidase and [beta]-glucosidase activities in short- and long-term incubations. Enzyme activity was independent of nutrient availability and suggested that microbial communities were not nutrient-limited. Time-course assays of bacterial hydrolysis of TOC, TN, and carbohydrates provided information about how substrate limitation may affect enzyme activity. Positive correlations between bulk TOC and TN content and enzyme activity indicated enzyme dependence on polymeric substrate content. Induction of enzyme activity after sediment enrichments with specific labile compounds confirmed the importance of available organic substrate to enzyme hydrolysis efficiency. A kinetic approach established the occurrence of enzyme inhibition and its effects on enzyme hydrolytic capacity. The addition of a specific-enzyme substrate to sediment samples modified enzyme parameters and indicated that a substrate-reversible type of inhibitor could reduce enzyme hydrolytic capacity. The addition of polyphenol, as a natural inhibitor of enzyme activity, to the sediment resulted in a concomitant reduction of leucine aminopeptidase activity and ammonium regeneration rate, and thus demonstrated a close coupling between enzyme activity and sediment ammonium regeneration. These research results demonstrate the dynamic nature of the hydrolytic enzymes, provide information about the mechanisms of induction and inhibition of activity, and demonstrate some implications of reducing the hydrolytic capacity to organic matter decomposition and nutrient regeneration rates. / text